It is known that bone morphogenetic proteins (BMPs) stimulate the healing of bone defects in mammals or primates, and particularly are secreted in bone cells to induce bone formation through receptors present in the adjacent cell membranes, when used with collagen or biodegradable polymers in demineralization conditions. The hBMPs are a group of proteins having similarity to each other, and are known to have more than 14 members, including hBMP2 through hBMP15 until now. The hBMP2, 3, 4, 6, 7 and 14 are known to have the medical efficacy of inducing bone regeneration.
Among them, the hBMP2 has been known to be the most effective bone morphogenetic protein. There have been a lot of studies on the medical effects and applications of BMP proteins. The BMP7 is known to not only suppress the fibrosis of organs by antagonistic action of TGF-β1, but also induce the regeneration of organs (Nature Med., 9:964, 2003; J. Biol. Chem., 280:8094, 2005). It is known that the BMP14, (GDF-5, growth/differentiation factor-5: MP-52) shows the function of effectively healing skin wounds and aiding the healing of gastric or duodenal ulcer when administered to human beings or mammals (U.S. Pat. No. 6,764,994; Nature, 368:639, 1994; Exp. Cell Res., 235:218, 1997; Neurosurg Focus, 13:1, 2002; U.S. Pat. No. 6,531,450). In the case of liver or kidney cirrhosis, the BMPs are also known to antagonize TGF-β to inhibit the formation of fibrous tissue and induce the recovery of normal tissue. Namely, the BMPs may function not only to stimulate the formation of bones or cartilages but also to regenerate the skin or regenerate gastrointestinal tissue, unlike the terminological definition of BMP.
As an old method for preparing BMPs, a method of extracting BMPs from demineralized animal bone tissue using natural salt (U.S. Pat. No. 4,294,753) has been attempted, but the method has problems in that the preparation efficiency is too low for mass production. A method was developed in early 1990s for separating and purifying the active recombinant hBMP2 after culturing CHO cells transformed with a BMP gene (Proc. Natl. Acad. Sci., 87:2220, 1990). Since then, the mass production of BMPs has been possible (U.S. Pat. Nos. 4,968,590; 5,106,626; 5,106,748; 5,166,058; 5,187,076; 5,187,623; 5,208,219; 5,258,494; 5,266,683; 5,284,756; 5,399,346; and 6,593,109).
However, the methods for preparing recombinant hBMP2 and hBMP7 using the transformed CHO cells have problems in that the culturing of a large amount of CHO cells is required to obtain enough amount of active rhBMP2 or rhBMP7. The separation and purification steps are very complicated, and the production cost is very high. Also, these methods have a common problem that the biological activity of the proteins is reduced during the separation, purification, storage, medication and/or administration processes. To improve a part of the shortcomings of these methods of preparing recombinant BMPs by culturing animal cells, Biopharm company recently developed a method for preparing rhBMP14(MP-52) by culturing recombinant E. coli at lower cost (US 2003/0181378; WO 96/33215). In this method of preparing the rhBMP14, however, the step of separating and purifying rhBMP14 in the form of an activated protein is still complicated and inconvenient. The problem of activity reduction of the prepared E. coli recombinant protein has still not been solved for the separation, purification, storage, handling, and/or administration steps.
Moreover, in producing active BMPs in recombinant E. coli as in the case of the prior Biopharm's method, there is a limitation on the selection and designing of the biochemical structures of BMPs. Even if it is possible to prepare a protein having the structure like rhBMP14, using the Biopharm's method, it shall not be possible to practically prepare the BMPs having similar biochemical structures like active rhBMP2, rhBMP4, rhBMP7, etc.
In the case of rhBMP-2, which is commercially available and is medically used for, e.g., spine fusion, it is being sold for as high as several thousand US dollars per mg in the year 2005. Despite that the rhBMPs have various latent potencies for numerous patients who need spine fusion operations, or who need regenerating gastric ulcer or liver cirrhosis, the clinical application of BMPs has been limited because of the extremely high cost and the inconveniences and activity loss in the storage, handling and administration.
Accordingly, in the art, there has been an urgent and strong need for the development of new kinds of biochemical drug substances which would provide equal or higher biomedical efficacy than those of rhBMPs or TGF-βs, and which can be produced at significantly lower costs than those for previously known proteins, and which can fundamentally solve the inconveniences and the activity reduction problem in the separation, purification, storage, handling, and administration steps.
Accordingly, the present inventors have spent extensive time and efforts to develop a group of new polypeptides, which can fundamentally solve the problems in the previously known activated proteins produced for stimulating the formation of bones or cartilages, or the regeneration of biological tissues including the skin wounds, ulcer, and/or liver cirrhosis. The new polypeptides have new biochemical structures and provide biomedical efficacy by new pharmacological mechanisms. The new kind of polypeptides can be directly medicated to human patients to stimulate the formation or regeneration of bones or cartilages or to improve or regenerate the fibrosis or cirrhosis of organs, such as kidneys, liver, lungs and heart, by new pharmacological mechanisms. The newly developed polypeptides are kept in a non-activated state in steps of the separation, purification, storage, and handling before administration. The new polypeptides are designed so that they shall be activated in vivo after administration to patients.
Namely, the present inventors have developed a new group of non-activated tissue-regeneration polypeptides (TRPs) containing: a protein transduction domain (PTD) making the proteins to permeate cell membranes; a furin activation domain (FAD) which has at least one proprotein convertase cleavage site and which can be cleaved by the proprotein convertase and can activate a non-activated tissue regeneration domain (TRD) in cells; and a non-activated TRD which can be activated by the proprotein convertase cleavage of the FAD. The in vivo activated polypeptides are designed to stimulate the growth or formation of tissue or to induce the regeneration of tissue. Also, the present inventors have found that the production cost of the present TRPs are significantly lower than those of the existing active proteins such as rhBMPs. It is much easier to store, handle, and administer the present TRPs than the existing rhBMPs. The present TRPs provide biomedical efficacies by new pharmacological mechanisms. Specifically the TRPs permeate cell membranes without help from receptors. They are cleaved by furin in cells and then activated. The activated proteins are secreted out of the cells in large amount, and then they stimulate cell membrane receptors for bone formation or tissue regeneration. On the basis of these new findings, the present invention has been completed.